Testing soil nematode extraction efficiency using different variations of the Baermann-funnel method
Keywords:soil organisms, comparability, reproducibility, extraction methods
Nematodes are increasingly used as powerful bioindicators of soil food web composition and functioning in ecological studies. Todays’ ecological research aims to investigate not only local relationships but global patterns, which requires consistent methodology across locations. Thus, a common and easy extraction protocol of soil nematodes is needed. In this study, we present a detailed protocol of the Baermann-funnel method and highlight how different soil pre-treatments and equipment (soil type, soil height, sieving, and filter type) can affect extraction efficiency and community composition by using natural nematode communities. We found that highest nematode extraction efficiency was achieved using lowest soil height as indicated by the thickness of the soil sample in the extractor due to differences in soil weight (1, 2, or 4 cm soil height) in combination with soil sieving (instead of no sieving), and by using milk filters (instead of paper towels). PCA at the family level revealed that different pre-treatments significantly affected nematode community composition. Increasing the height of the soil sample by adding more soil increased the proportion of larger-sized nematodes likely because those are able to overcome long distances but selected against small nematodes. Sieving is suggested to break up soil aggregates and, therefore, facilitate moving in general. Interestingly, sieving did not negatively affect larger nematodes that are supposed to have a higher probability of getting bruised during sieving but, even if not significant, yielded more extracted nematodes than no sieving. We therefore recommend to use small heights of sieved soil with milk filter to extract free-living soil nematodes with the Baermann-funnel method. The present study shows that variations in the extraction protocol can alter the total density and community composition of extracted nematodes and provides recommendations for an efficient and standardized approach in future studies. Having a simple, cheap, and standardized extraction protocol can facilitate the assessment of soil biodiversity in global contexts.
Ainsworth, R. & World Health Organization (2004): Safe Piped Water: Managing Microbial Water Quality in Piped Distribution Systems. – IWA Publishing, London: 168pp.
Andrássy, I. (2005): Free-living nematodes of Hungary, I, 3rd ed. – István Matskási, Budapest.
Baermann, G. (1917): Eine einfache Methode zur Auffindung von Anklostomum (Nematoden) Larven in Erdproben. – Tijdschr Diergeneeskd 57: 131–137.
Bongers, T. (1994): De nematoden van Nederland, 2nd ed. – KNNV, Utrecht.
Bongers, T. (1990): The maturity index: an ecological measure of environmental disturbance based on nematode species composition. – Oecologia 83: 14–19.
Bongers, T. & M. Bongers (1998): Functional diversity of nematodes. – Applied Soil Ecology 10: 239–251.
Bongers, T. & H. Ferris (1999): Nematode community structure as a bioindicator in environmental monitoring. – Trends in Ecology & Evolution 14: 224–228.
Borer, E. T., W. S. Harpole, P. B. Adler, E. M. Lind, J. L. Orrock, E. W. Seabloom & M. D. Smith (2014): Finding generality in ecology: A model for globally distributed experiments. – Methods in Ecology and Evolution 5: 65–73.
Bruelheide, H., K. Nadrowski, T. Assmann, J. Bauhus, S. Both, F. Buscot, X.-Y. Chen, B. Ding, W. Durka, A. Erfmeier, J. L. M. Gutknecht, D. Guo, L.-D. Guo, W. Härdtle, J.-S. He,
A.-M. Klein, P. Kühn, Y. Liang, X. Liu, S. Michalski, P. A. Niklaus, K. Pei, M. Scherer-Lorenzen, T. Scholten, A. Schuldt, G. Seidler, S. Trogisch, G. von Oheimb, E. Welk, C. Wirth,
T. Wubet, X. Yang, M. Yu, S. Zhang, H. Zhou, M. Fischer,
K. Ma & B. Schmid (2014): Designing forest biodiversity experiments: general considerations illustrated by a new large experiment in subtropical China. – Methods in Ecology and Evolution 5: 74–89.
Cesarz, S., M. Ciobanu, A. J. Wright, A. Ebeling, A. Vogel, W. W. Weisser & N. Eisenhauer (2017): Plant species richness sustains higher trophic levels of soil nematode communities after consecutive environmental perturbations. – Oecologia 184: 715–728.
Cesarz, S., P. B. Reich, S. Scheu, L. Ruess, M. Schaefer & N. Eisenhauer (2015): Nematode functional guilds, not trophic groups, reflect shifts in soil food webs and processes in response to interacting global change factors. – Pedobiologia 58: 23–32.
Delgado-Baquerizo, M., A. M. Oliverio, T. E. Brewer, A. Benavent-González, D. J. Eldridge, R. D. Bardgett, F. T. Maestre, B. K. Singh & N. Fierer (2018): A global atlas of the dominant bacteria found in soil. – Science 359: 320–325.
Eisenhauer, N. & C. A. Guerra (2019): Global maps of soil-dwelling nematode worms. – Nature [https://doi.org/10.1038/d41586-019-02197-0].
Gingold, R., T. Moens & A. Rocha-Olivares (2013): Assessing the Response of Nematode Communities to Climate Change-Driven Warming: A Microcosm Experiment. – PLoS ONE 8.
Hantsch, L., S. Bien, S. Radatz, U. Braun, H. Auge & H. Bruelheide (2014): Tree diversity and the role of non-host neighbour tree species in reducing fungal pathogen infestation. – The Journal of Ecology 102: 1673–1687.
Keuskamp, J. A., B. J. J. Dingemans, T. Lehtinen, J. M. Sarneel & M. M. Hefting (2013): Tea Bag Index: A novel approach to collect uniform decomposition data across ecosystems. – Methods in Ecology and Evolution 4: 1070–1075.
MacMillan, K., S. Haukeland, R. Rae, I. Young, J. Crawford, S. Hapca & M. Wilson (2009): Dispersal patterns and behaviour of the nematode Phasmarhabditis hermaphrodita in mineral soils and organic media. – Soil Biology and Biochemistry 41: 1483–1490.
Meyl, A. (1961): Fadenwürmer (Nematoden). – Franckh’sche Verlagshandlung, Stuttgart.
Nielsen, U. N., E. Ayres, D. H. Wall, G. Li, R. D. Bardgett, T. Wu & J. R. Garey (2014): Global-scale patterns of assemblage structure of soil nematodes in relation to climate and ecosystem properties. – Global Ecology and Biogeography 23: 968–978.
Orgiazzi, A., R. D. Bardgett, E. Barrios, V. Behan-Pelletier,
M. J. I. Briones, J.-L. Chotte, G .B. De Deyn, P. Eggleton,
N. Fierer, T. Fraser, K. Hedlund, S. Jeffery, N. C. Johnson,
A. Jones, E. Kandeler, N. Kaneko, P. Lavelle, & M. Lemanceau (2016): Global soil biodiversity atlas. – Publications Office of the European Union, Luxembourg.
Pei, Z. Q., K. N. Leppert, D. Eichenberg, H. Bruelheide, P. A. Niklaus, F. Buscot & J. L. M. Gutknecht (2017): Leaf litter diversity alters microbial community structure and nutrient cycling in a subtropical forest ecosystem. – Biogeochemistry 134: 163–181.
Phillips, H. R. P., E. K. Cameron, O. Ferlian, M. Türke, M. Winter & N. Eisenhauer (2017): Red list of a black box. – Nature Ecology & Evolution 1: 0103.
PM7/119(1) (2013): Nematode extraction. – EPPO Bulletin 43: 471–495.
R Development Core Team (2008): R: A language and environment for statistical computing. – R Foundation for Statistical Computing. Vienna, Austria.
Ramirez, K. S., C. G. Knight, M. de Hollander, F. Q. Brearley, B. Constantinides, A. Cotton, S. Creer, T. W. Crowther,
J. Davison, M. Delgado-Baquerizo, E. Dorrepaal, D. R. Elliott, G. Fox, R. I. Griffiths, C. Hale, K. Hartman, A. Houlden, D. L. Jones, E. J. Krab, F. T. Maestre, K. L. McGuire, S. Monteux, C. H. Orr, W. H. van der Putten, I. S. Roberts, D. A. Robinson, J. D. Rocca, J. Rowntree, K. Schlaeppi, M. Shepherd, B. K. Singh, A. L. Straathof, J. M. Bhatnagar, C. Thion, M. G. A. van der Heijden & F. T. de Vries (2018): Detecting macroecological patterns in bacterial communities across independent studies of global soils. – Nature Microbiology 3: 189–196.
Robinson, A. F. & C. M. Heald (1989): Accelerated movement of nematodes from soil in baermann funnels with temperature gradients. – Journal of Nematology 21: 370–378.
Roscher, C., J. Schumacher, J. Baade, W. Wilcke, G. Gleixner, W. W. Weisser, B. Schmid, E. Schulze, I. Ökologie, F. Jena & I. Geographie (2004): The role of biodiversity for element cycling and trophic interactions : an experimental approach in a grassland community. – Basic and Applied Ecology 121: 107–121.
Ruess, L. (1995): Studies on the nematode fauna of an acid forest soil: spatial disturbance and extraction. – Nematologica 41: 229–239.
Song, D., K. Pan, A. Tariq, F. Sun, Z. Li & X. Sun (2017): Large-scale patterns of distribution and diversity of terrestrial nematodes. – Applied Soil Ecology.
Tedersoo, L., M. Bahram, S. Põlme, U. Kõljalg, N. S. Yorou, R. Wijesundera, L. V. Ruiz, A. M. Vasco-Palacios, P. Q. Thu, A. Suija, M. E. Smith, C. Sharp, E. Saluveer, A. Saitta, M. Rosas, T. Riit, D. Ratkowsky, K. Pritsch, K. Põldmaa, M. Piepenbring, C. Phosri, M. Peterson, K. Parts, K. Pärtel, E. Otsing, E. Nouhra, A. L. Njouonkou, R. H. Nilsson, L. N. Morgado, J. Mayor, T. W. May, L. Majuakim, D. J. Lodge, S. S. Lee, K.-H. Larsson, P. Kohout, K. Hosaka, I. Hiiesalu, T. W. Henkel, H. Harend, L. Guo, A. Greslebin, G. Grelet, J. Geml, G. Gates, W. Dunstan, C. Dunk, R. Drenkhan, J. Dearnaley, A. De Kesel, T. Dang, X. Chen, F. Buegger, F. Q. Brearley, G. Bonito, S. Anslan, S. Abell & K. Abarenkov (2014): Global diversity and geography of soil fungi. – Science 346: 1256688.
van Bezooijen, J. (2006): Methods and techniques for nematology. – Wageningen.
van den Hoogen, J., S. Geisen, D. Routh, H. Ferris, W. Traunspurger, D. A. Wardle, R. G. M. de Goede, B. J. Adams, W. Ahmad, W. S. Andriuzzi, R. D. Bardgett, M. Bonkowski, R. Campos-Herrera, J. E. Cares, T. Caruso, L. de Brito Caixeta, X. Chen, S. R. Costa, R. Creamer, J. Mauro da Cunha Castro, M. Dam, D. Djigal, M. Escuer, B. S. Griffiths, C. Gutiérrez, K. Hohberg, D. Kalinkina, P. Kardol, A. Kergunteuil, G. Korthals, V. Krashevska, A. A. Kudrin, Q. Li, W. Liang, M. Magilton, M. Marais, J. A. R. Martín, E. Matveeva, El Hassan Mayad, C. Mulder, P. Mullin, R. Neilson, T. A. Duong Nguyen, U. N. Nielsen, H. Okada, J. E. Palomares Rius, K. Pan, V. Peneva, L. Pellissier, J. C. Pereira da Silva, C. Pitteloud, T. O. Powers, K. Powers, C. W. Quist, S. Rasmann, S. Sánchez Moreno, S. Scheu, H. Setälä, A. Sushchuk, A. V. Tiunov, J. Trap, W. van der Putten, M. Vestergård, C. Villenave, L. Waeyenberge, D. H. Wall, R. Wilschut, D. G. Wright, J. Yang & T. Ward Crowther (2019): Soil nematode abundance and functional group composition at a global scale. – Nature [https://doi.org/10.1038/s41586-019-1418-6].
Van Voorhies, W. A & S. Ward, S. (2000): Broad oxygen tolerance in the nematode Caenorhabditis elegans. – The Journal of Experimental Biologyxperimental Biology 203: 2467–2478.
Verheyen, K., M. Vanhellemont, H. Auge, L. Baeten, C. Baraloto, N. Barsoum, S. Bilodeau-Gauthier, H. Bruelheide, B. Castagneyrol, D. Godbold, J. Haase, A. Hector, H. Jactel, J. Koricheva, M. Loreau, S. Mereu, C. Messier, B. Muys, P. Nolet, A. Paquette, J. Parker, M. Perring, Q. Ponette, C. Potvin, P. Reich, A. Smith, M. Weih & M. Scherer-Lorenzen (2015): Contributions of a global network of tree diversity experiments to sustainable forest plantations. – Ambio.
Verschoor, B. & R. G. M. De Goede (2000): The nematode extraction efficiency of the Oostenbrink elutriator-cottonwool filter method with special reference to nematode body size and life strategy. – Nematology 2: 325–342.
Viglierchio, D. R. & R. V. Schmitt (1983): On the methodology of nematode extraction from field samples: comparison of methods for soil extraction. – Journal of Nematology 15: 450–454.
Viketoft, M. & B. Sohlenius (2011): Soil nematode populations in a grassland plant diversity experiment run for seven years. – Applied Soil Ecology 48: 174–184.
Wallace, H. R. (1968): The dynamics of nematode movement. – Annual Review of Phytopathology 6: 91–114.
Yeates, G. W. (1999): Effects of plants on nematode community structure. – Annual Review of Phytopathology 37: 127–149.
Yeates, G. W., T. Bongers, R. G. M. de Goede, D. W. Freckman & S. S. Georgieva (1993): Feeding habits in soil nematode families and genera - an outline for soil ecologist. – Journal of Nematology 25: 315–331.